Multidrug resistant (MDR) Gram-negative bacterial infections are a serious threat to human health due to the lack of effective treatments. In this study, we selected 50 Gram-negative bacterial strains, including 26 strains of Klebsiella pneumoniae and 24 strains of Escherichia coli, to explore whether resveratrol and polymyxin B have a synergistic killing effect. MIC values against polymyxin B were ≥ 4 μg/mL for 44 of the strains and were 2 μg/mL for the other 6 strains. MICs against polymyxin B in the isolates tested were significantly reduced by the addition of resveratrol. The degree of decline depended on the bacteria, ranging from 1/2 MIC to 1/512 MIC, and the higher the concentration of resveratrol, the greater the decrease. Checkerboard analysis indicated a synergistic effect between resveratrol and polymyxin B; the optimal drug concentration for different bacteria was different, that of resveratrol ranging from 32 μg/mL to 128 μg/mL. Subsequent time-kill experiments showed that a combination of polymyxin B and resveratrol was more effective in killing bacteria. Our in vitro studies have shown that resveratrol can increase the sensitivity of MDR bacterial strains to polymyxin B, suggesting a potential new approach to the treatment of MDR infections.